Antibodies specific for antigens that are integrated into the cell membrane (e.g. receptors) are a valuable tool in research and clinical application. The application of many standard procedures for high-throughput screening of such antibodies is hampered by the fact that the protein has to be used apart from its natural environment, the plasma membrane. Usually, the soluble portion of membrane proteins are expressed recombinantly and applied in screening assays. It is therefore more desirable to screen for antibodies that interact with the antigen that is integrated in the membrane.
Fluorescence-activated cell-sorting (FACS) is a specialized type of flow cytometry and provides a method for sorting cells or analyzing the functional interaction of cells with a variety of molecules including antibodies. FACS methods or other currently used technologies (e.g. immunofluorescence microscopy) are time consuming and thus not optimal for a high-throughput screening during a cell selection process. These methods are usually applied at the end of the selection process to verify the quality of e.g. an antibody. During the selection process of cell lines expressing recombinant antibodies the cells are routinely screened only for their productivity by measuring the amount of expressed antibodies. In the worst case this could finally lead to a cell line expressing high amounts of antibodies which may not have the expected functionality, i.e. that the antibodies do not bind to the respective antigen (membrane-protein) in it's natural environment, the cell membrane This may be especially the case if the antibody to be recombinantly expressed is not well characterized prior to expression. It would be therefore advantageous to screen not only for high expression during the selection process but also for the binding of the required antibody to the membrane-protein in the cell membrane.
One typical example for this problem is the murine antibody secreted by the mouse hybridoma cell line 9C5/9069. It binds to the human cluster designation 34 (CD34) antigen and can be used for the isolation of CD34 positive stem cells. The 9C5/9069 mouse hybridoma was originally developed by Lansdorp et al. (Leucocyte Typing IV, 1989; 826-7, Oxford University Press, Oxford). There is an established production process for said antibody using the hybridoma cell line. However, the current process faces some limitations regarding the upscale of production and improvement of production yield. In addition the antibody is not characterized in detail, i.e. the sequence is not known.
In deciding for a production cell line expressing anti-CD34 antibody, one parameter is an acceptable cell-specific average productivity (determined e.g. as picogram antibody produced per cell per day, pcd) and sufficient yield in the cell culture, measured e.g. as mg/L of antibody. These parameters are usually determined by standard ELISA methods.
The anti-CD34 antibodies are routinely used for the selection of stem cells and there are clinical applications of said selected stem cells for a variety of diseases (Kawabata et al. Ther Apher Dial. 2003; 7:298-304).    EP1083226A1 discloses devices containing antibodies recognizing CD4 or CD34 and their use for the separation of CD4 or CD34 positive cells.    EP0765478B1 teaches to select CD34 target cells by reacting specific anti-CD34 antibody to surface antigen and then disrupting the complex formed with peptide displacer.    EP0695346A1 discloses the separation of hematopoietic progenitor cells on the basis of binding to a specific cell surface antigen such as CD34.
Interaction of ligands (e.g. antibodies) with cell surface proteins are often detected by growing adherent cells in wells, fixation of the cells and incubating them with the respective labeled ligand. These methods usually suffer from the fact that fixation results in (partial) denaturation of the cell surface proteins and thus might lead to falsified results.
However, none of these disclosures teaches a high-throughput assay for the selection of anti-CD34 antibodies that recognize the CD34 membrane-protein in it's natural environment, the cell membrane.
Therefore it was the inventive task of the present invention to develop a novel assay allowing to test the binding of antibodies to proteins in it's natural environment in a high-throughput format, preferably on microplates.